The ability to detect differences in DNA sequence (i.e. mutations) is of great importance in the field of medical genetics. For example, the detection of mutations directly in genomic DNA is essential for identifying polymorphisms for genetic studies, to determine the molecular basis of inherited diseases and to provide carrier and prenatal diagnosis for genetic counselling. Traditionally, detection of DNA variation has been performed by analysis of RFLPs using the Southern blotting technique (Southern, EM J Mol Biol 98:503-517 (1975)): Kan Y and A Dozy Nature 313:369-374 (1978); Wyman, A. and R. White Proc Natl. Acad. Sci. USA 77:6754-6758 (1980)). However, as this approach is relatively slow and technically demanding, new methods based on the polymerase chain reaction have been developed. These include (RFLP) analysis (Chehab et. al. Nature 329:293-294 (1987), the creation of artificial RFLPs by the use of primer-specified restriction-map modification (Hallassos A. et. al. Nucleic Acids Res. 17:3606 (1989)), hybridization to allele-specific oligonucleotides (ASOs) (Saiki et. al. Nature 324:163-166 (1986)) or detection of small deletions by determination of the size of the PCR product (Rommens et. al. Am J. Hum Genet 46:395-396 (1990)). Of these methods, only the ASO approach can be used to detect any point mutation or small deletion, as the other methods are all dependent on the nature of the mutation and the surrounding DNA sequence.
It is now becoming clear that, for many genetic diseases, there is more than one mutation responsible for the condition. For example, to date more than 225 cystic fibrosis (CF) disease causing mutations have been reported (CF Genetic Analysis Consortium, unpublished data), while not accounting for all cases of CF. Furthermore, the mutations can be closely spaced often within a few base pairs of each other. Examples of multimutational diseases include CF (Cutting G. et. al. Nature 346:366-369 (1990)); .beta.-thalassaemia (Old JM et. al. Lancet 336:834-837 (1990)) Tay-Sachs disease (Myerowitz R. Proc Natl Acad Sci USA 85:3955-3959 (1988)) and Sickle cell anemia (Saiki, R. K. et. al. Science 230, 1350-1354 (1985)). The presence of multiple potential mutations makes the detection of these diseases complex.
A method which enables the simultaneous analysis of a sample for the presence of multiple mutations would be useful.